Methods of inducing T cell non-responsiveness to transplanted tissues and of treating graft-versus-host-disease with anti-gp39 antibodies

ABSTRACT

Antibodies that bind a protein gp39 (also referred to as CD40 ligand) are disclosed. Preferably, the antibodies are monoclonal antibodies of an IgG1 isotype and bind human gp39. In a preferred embodiment, an antibody of the invention binds an epitope recognized by a monoclonal antibody 24-31, produced by a hybridoma 24-31 (ATTC Accession No. HB11712) or binds an epitope recognized by a monoclonal antibody 89-76, produced by a hybridoma 89-76 (ATCC Accession No. HB 11713). Pharmaceutical compositions comprising the antibodies of the invention are also disclosed. The antibodies of the invention are useful for inhibiting B cell proliferation and differentiation, T cell responses and for inducing T cell tolerance. Nucleic acid molecules encoding anti-gp39 antibodies, or portions thereof, as well as expression vectors and host cells incorporating said nucleic acid molecules, are also encompassed by the invention.

GOVERNMENT FUNDING

The work leading to this invention may have been supported by one ormore grants from the U.S. government. The U.S. government therefore mayhave certain rights in this invention

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 08/475,847,filed Jun. 7, 1995 now U.S. Pat. No. 5,747,037, in turn acontinuation-in-part of application Ser. No. 08/232,929, filed Apr. 25,1994, in turn a continuation-in-part of application Ser. No. 08/116,255,filed Sep. 2, 1993, now abandoned.

BACKGROUND OF THE INVENTION

To induce antigen-specific T cell activation and clonal expansion, twosignals provided by antigen-presenting cells (APCs) must be delivered tothe surface of resting T lymphocytes (Jenkins, M. and Schwartz, R.(1987) J Exp. Med. 165, 302-319; Mueller, D. L., et al. (1990) J.Immunol. 144, 3701-3709; Williams, I. R. and Unanue, E. R. (1990) JImmunol 145, 85-93). The first signal, which confers specificity to theimmune response, is mediated via the T cell receptor (TCR) followingrecognition of foreign antigenic peptide presented in the context of themajor histocompatibility complex (MHC). The second signal, termedcostimulation, induces T cells to proliferate and become functional(Schwartz, R. H. (1990) Science 248, 1349-1356). Costimulation isneither antigen-specific, nor MHC restricted and is thought to beprovided by one or more distinct cell surface molecules expressed byAPCs (Jenkins, M. K., et al. (1988) J. Immunol. 140, 3324-3330; Linsley,P. S., et al. (1991) J. Exp. Med. 173, 721-730, Gimmi, C. D., et al.,(1991) Proc. Natl. Acad. Sci. USA. 88, 6575-6579; Young, J. W., et al.(1992) J. Clin. Invest. 90, 229-237; Koulova, L., et al. (1991) J. Exp.Med 173, 759-762; Reiser, H., et al. (1992) Proc. Natl. Acad. Sci. USA.89, 271-275; van-Seventer, G. A., et al. (1990) J. Immunol. 144,4579-4586; LaSalle, J. M., et al., (1991) J. Immunol. 147, 774-80;Dustin, M. I., et al., (1989) J. Exp. Med. 169, 503; Armitage, R. J., etal. (1992) Nature 357, 80-82; Liu, Y., et al. (1992) J. Exp. Med. 175,437-445). One costimulatory pathway involved in T cell activationinvolves the molecule CD28 on the surface of T cells. This molecule canreceive a costimulatory signal delivered by a ligand on B cells or otherAPCs. Ligands for CD28 include members of the B7 family of B lymphocyteactivation antigens, such as B7-1 and/or B7-2 (Freedman, A. S. et al.(1987) J. Immunol. 137, 3260-3267; Freeman, G. J. et al. (1989) J.Immunol. 143, 2714-2722; Freeman, G. J. et al. (1991) J. Exp. Med 174,625-631; Freeman, G. J. et al. (1993) Science 262, 909-911; Azuma, M. etal. (1993) Nature 366, 76-79; Freeman, G. J. et al. (1993) J. Exp. Med.178, 2185-2192). B7-1 and B7-2 are also ligands for another molecule,CTLA4, present on the surface of activated T cells, although the role ofCTLA4 in costimulation is unclear.

Delivery of an antigen-specific signal with a costimulatory signal to aT cell leads to cell activation, which can include both T cellproliferation and cytokine secretion. In contrast, delivery of anantigen-specific signal to a T cell in the absence of a costimulatorysignal is thought to induce a state of unresponsiveness or anergy in theT cell, thereby inducing antigen-specific tolerance in the T cell.

Interactions between T cells and B cells play a central role in immuneresponses. Induction of humoral immunity to thymus-dependent antigensrequires "help" provided by T helper (hereafter Th) cells. While somehelp provided to B lymphocytes is mediated by soluble molecules releasedby Th cells (for instance lymphokines such as IL-4 and IL-5), activationof B cells also requires a contact-dependent interaction between B cellsand Th cells. Hirohata et al., J. Immunol., 140:3736-3744 (1988);Bartlett et al., J. Immunol., 143:1745-1754 (1989). This indicates thatB cell activation involves an obligatory interaction between cellsurface molecules on B cells and Th cells. The molecule(s) on the T celltherefore mediates contact-dependent helper effector functions of Tcells. A contact-dependent interaction between molecules on B cells andT cells is further supported by the observation that isolated plasmamembranes of activated T cells can provide helper functions necessaryfor B cell activation. Brian, Proc. Natl. Acad. Sci. USA, 85:564-568(1988); Hodgkin et al., J. Immunol., 145:2025-2034 (1990); Noelle etal., J. Immunol., 146:1118-1124 (1991).

A molecule, CD40, has been identified on the surface of immature andmature B lymphocytes which, when crosslinked by antibodies, induces Bcell proliferation. Valle et al., Eur. J. Immunol., 19:1463-1467 (1989);Gordon et al., J. Immunol., 140:1425-1430 (1988); Gruber et al., J.Immunol., 142: 4144-4152 (1989). CD40 has been molecularly cloned andcharacterized. Stamenkovic et al., EMBO J., 8:1403-1410 (1989). A ligandfor CD40, gp39 (also called CD40 ligand or CD40L) has also beenmolecularly cloned and characterized. Armitage et al., Nature, 357:80-82(1992); Lederman et al., J. Exp. Med., 175:1091-1101 (1992); Hollenbaughet al., EMBO J., 11:4313-4319 (1992). The gp39 protein is expressed onactivated, but not resting, CD4⁺ Th cells. Spriggs et al., J. Exp. Med.,176:1543-1550 (1992); Lane et al., Eur. J. Immunol., 22:2573-2578(1992); Roy et al., J. Immunol., 151:1-14 (1993). Cells transfected withthe gp39 gene and expressing the gp39 protein on their surface cantrigger B cell proliferation and, together with other stimulatorysignals, can induce antibody production. Armitage et al., Nature,357:80-82 (1992); Hollenbaugh et al., EMBO J., 11:4313-4319 (1992).

SUMMARY OF THE INVENTION

Cell-surface molecules which mediate contact-dependent helper effectorfunctions of T cells are important for inducing immune responses whichrequire T cell help. For example, the interaction of gp39 on T cellswith CD40 on B cells plays a central role in activating B cell responsesto antigens. The current invention is based, at least in part, on thediscovery that cell-surface molecules which mediate contact-dependenthelper effector functions also play a critical role in the response of Tcells to antigens. In particular, it has been discovered that, underappropriate conditions, interference of an interaction between gp39 on aT cell and a ligand on a cell which is presenting antigen to the T cellcan induce antigen-specific T cell tolerance. Accordingly, the cellwhich presents antigen to the T cell requires an interaction between agp39 ligand (e.g., CD40) on the cell and gp39 on the T cell to be ableto provide signals necessary for activation of the T cell. Inhibition ofthe interaction between the gp39 ligand and gp39 prevents T cellactivation and rather induces antigen-specific T cell tolerance.

The methods of the invention pertain to induction of antigen-specific Tcell tolerance. The methods involve contacting a T cell with: 1) a cellwhich presents antigen to the T cell and has a ligand on the cellsurface which interacts with a receptor on the surface of the T cellwhich mediates contact-dependent helper effector functions; and 2) anantagonist of the receptor on the surface of a T cell which mediatescontact-dependent helper effector functions. The antagonist inhibits theinteraction of the receptor with it's ligand. A T cell can be contactedwith the cell which presents antigen and the antagonist in vitro, oralternatively, the cell and the antagonist can be administered to asubject to induce T cell tolerance in vivo.

In a preferred embodiment, the receptor on the surface of the T cellwhich mediates contact-dependent helper effector functions is gp39. Inthis embodiment, the antagonist is a molecule which inhibits theinteraction of gp39 with its ligand on a cell which presents antigen tothe T cell. A particularly preferred gp39 antagonist is an anti-gp39antibody. Alternatively, the gp39 antagonist is a soluble form of a gp39ligand, for example soluble CD40. The cell which presents antigen to a Tcell is preferably a B cell. The B cell can be a small, resting B cell.To induce T cell tolerance to a soluble antigen, the B cell can becontacted with the antigen prior to contact with the T cell (e.g., priorto administration to a subject). In another embodiment, to induce T celltolerance to alloantigens, the cell which is used to present antigen tothe T cell is an allogeneic cell. The allogeneic cell can be, forexample, an allogeneic B cell, allogeneic bone marrow, allogeneic spleencells or allogeneic cells in peripheral blood.

The methods of the current invention can be used, for example, to induceT cell tolerance to a soluble antigen, to induce T cell tolerance a bonemarrow transplant or other organ transplant or to inhibitgraft-versus-host disease in bone marrow transplantation. In the case ofbone marrow transplantation, the transplanted bone marrow cellsthemselves serve as cells which present antigen to the T cell in themethod of the invention. Accordingly, in one embodiment of theinvention, acceptance of a bone marrow transplant is promoted byadministering to a subject allogeneic bone marrow in conjunction with agp39 antagonist (e.g., an anti-gp39 antibody).

This invention further pertains to anti-human gp39 monoclonal antibodiescapable of inhibiting B cell proliferation, B cell differentiation and Tcell responses and to pharmacuetical compositions comprising suchantibodies. Anti-human gp39 monoclonal antibodies of the invention arepreferred for use in modulating immune responses in general, andparticularly for use in inducing antigen-specific T cell tolerance.Preferred antibodies include monoclonal antibodies 3E4, 2H5, 2H8, 4D9-8,4D9-9, 24-31, 24-43, 89-76 and 89-79, described in Example 6.Particularly preferred antibodies are monoclonal antibodies 89-76 and24-31. The 89-76 and 24-31 hybridomas, producing the 89-76 and 24-31antibodies, respectively, were deposited under the provisions of theBudapest Treaty with the American Type Culture Collection, ParklawnDrive, Rockville, Md., on Sep. 2, 1994. The 89-76 hybridoma was assignedATCC Accession Number HB11713 and the 24-31 hybridoma was assigned ATCCAccession Number HB11712. The 24-31 and 89-76 antibodies are of the IgG1isotype.

Accordingly, in one embodiment, the invention provides an anti-humangp39 monoclonal antibody (mAb) of an IgG1 isotype. The anti-human gp39mAb of the invention can inhibit B cell proliferation in a standard invitro assay, for example, B cell proliferation induced by treatment ofthe B cells with interleukin-4 and soluble gp39. Preferably, theanti-human gp39 antibody inhibits B cell proliferation with an IC₅₀(i.e., concentration necessary to inhibit proliferation by 50%) betweenabout 0.01 and 5.0 μg/ml, more preferably between about 0.1 and 2.5μg/ml, and even more preferably between about 0.1 and 1.25 μg/ml. Theanti-human gp39 mAbs of the invention can also inhibit B cell productionof IgG, IgM and/or IgA in a standard in vitro assay, for example, Igproduction induced by culturing of B cells with activated T cells (e.g.,T cells activated by treatment with anti-CD3 antibody). Preferably, theanti-human gp39 antibody inhibits B cell production of IgG, IgM and/orIgA with an IC50 between about 0.01 and 1.0 μg/ml or, more preferably,between about 0.01 and 0.1 μg/ml.

In a preferred embodiment, the anti-human gp39 mAb of the inventionbinds an epitope recognized by a monoclonal antibody selected from agroup consisting of 3E4, 2H5, 2H8, 4D9-8, 4D9-9, 24-31, 2443, 89-76 and89-79. More preferably, the anti-human gp39 mAb binds an epitoperecognized by monoclonal antibody 24-31 or monoclonal antibody 89-76.The ability of an mAb to bind an epitope recognized by any of theaforementioned antibodies can be determined by standardcross-competition assays. For example, an antibody that binds the sameepitope recognized by mAb 24-31 will compete for the binding of labeled24-31 to activated T cells, whereas an antibody that binds a differentepitope than that recognized by mAb 24-31 will not compete for thebinding of labeled 24-31 to activated T cells.

The invention also provides pharmaceutical compositions of theanti-human gp39 antibodies of the invention. These compositionstypically comprise an anti-human gp39 mAb (e.g., preferably 24-31 or89-76) and a pharmaceutically acceptable carrier.

Yet another aspect of the invention pertains to nucleic acid encoding ananti-human gp39 mAb (e.g., DNA encoding an immunoglobulin heavy chain orlight chain, or portion thereof, of an anti-human gp39 mAb). Suchnucleic acid can be isolated from a cell (e.g., hybridoma) producing ananti-human gp39 mAb by standard techniques. For example, nucleic acidencoding the 24-31 or 89-76 mAb can be isolated from the 24-31 or 89-76hybridoma, respectively, by cDNA library screening, PCR amplification orother standard technique. Nucleic acid encoding an anti-human gp39 mAbchain can be manipulated by standard recombinant DNA techniques toproduce recombinant anti-human gp39 mAbs, for example, chimeric orhumanized anti-human gp39 mAbs.

Moreover, nucleic acid encoding an anti-human gp39 mAb can beincorporated into an expression vector and introduced into a host cellto facilitate expression and production of recombinant forms ofanti-human gp39 antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic representation of T cell tolerance to a proteinantigen induced by in vivo anti-gp39 treatment. T cells responses weremeasured in vitro upon challenge with an antigen which previously wasadministered in vivo on antigen-pulsed B cells either with or without ananti-gp39 antibody.

FIG. 2 is a graphic representation of T cell tolerance to allogeneic Bcells induced by in vivo anti-gp39 treatment. T cells responses weremeasured in vitro upon challenge with allogeneic B cells whichpreviously were administered in vivo either with or without an anti-gp39antibody.

FIG. 3A is a graphic representation of the inhibition of primaryallogeneic CTL responses induced by allogeneic B cells when recipientanimals are treated with anti-gp39 antibody. Groups represented areuntreated mice (▪), anti-gp39 treated mice (Δ) and spleen cells fromunprimed Balb/c mice (; used as negative control effector cells).

FIGS. 3B and 3C are graphic representations of the inhibition of primaryallogeneic CTL responses induced by LPS-treated B cell blasts whenrecipient animals are treated with anti-gp39 antibody. Panels B and Crepresent two independent experiments. Groups represented are LPS blastsin vivo without treatment (>), LPS blasts in vivo with anti-gp39treatment (), resting B cells in vivo without treatment (◯) and restingB cells in vivo with anti-gp39 treatment (□).

FIG. 4A is a graphic representation of the inhibition of secondaryallogeneic CTL responses induced by allogeneic B cells when recipientanimals are treated with anti-gp39 antibody. Effector groups shown are:HIg treated recipients (), naive Balb/c (>) and anti-gp39 treatedrecipients (▪). Corresponding syngeneic response (Balb/c cellsstimulated with Balb/c cells) are indicated by open symbols.

FIG. 4B is a graphic representation of the specificity of inhibition ofallogeneic CTL responses by anti-gp39 treatment. Shown are CTL responsesagainst H-2^(k) targets by naive Balb/c cells (◯) or by cells tolerizedto H-2^(b) by administration of H-2^(b) haplotype B cells and anti-gp39(□).

FIG. 5 is a bar graph depicting the number of splenocytes in a hostwhich has received a bone marrow transplant at various times aftertransfer of the bone marrow to the host either with or without treatmentwith an anti-gp39 antibody.

FIG. 6A is a graphic representation of the concentration of IgA producedby splenic B cells in vitro after removal from mice which received abone marrow transplant either with or without in vivo anti-gp39treatment. Splenic B cells were removed and antibody production measuredeither 7 or 14 days after bone marrow transplantation.

FIG. 6B is a graphic representation of the concentration of IgG1produced by splenic B cells in vitro after removal from mice whichreceived a bone marrow transplant either with or without in vivoanti-gp39 treatment. Splenic B cells were removed and antibodyproduction measured either 7 or 14 days after bone marrowtransplantation.

FIG. 7A is a graphic representation of the serum IgE concentrations inmice which received a bone marrow transplant either with or without invivo anti-gp39 treatment at various times after bone marrow transfer.

FIG. 7B is a graphic representation of the serum anti-DNA antibodyconcentrations in mice which received a bone marrow transplant eitherwith or without in vivo anti-gp39 treatment at various times after bonemarrow transfer.

FIGS. 8A and 8B are graphic representations depicting the cytolyticactivity in vitro of cytotoxic T cells from mice which received a bonemarrow transplant either with or without in vivo anti-gp39 treatment atdifferent effector to target cell ratios (E:T ratio). Panels A and Brepresent two independent experiments.

FIGS. 9A, 9B and 9C are flow cytometic profiles depicting the stainingof 6 hour activated human peripheral blood lymphocytes with eitherCD40Ig (panel A), mAb 4D9-8 (panel B) or mAb 4D9-9 (panel C).

FIGS. 10A, 10B and 10C are flow cytometic profiles depicting thestaining of 6 hour activated human peripheral blood lymphocytes culturedin the presence of cycloporin A stained with either mAb 4D9-8 (panel A),mAb 4D9-9 (panel B) or CD40Ig (panel C).

FIGS. 11A and 11B are flow cytometric profiles depicting the staining of6 hour activated human peripheral blood lymphocytes with CD40Ig in thepresence of unlabeled mAb 4D9-8 (panel A) or unlabeled mAb 4D9-9 (panelB).

FIG. 12 is a graphic representation of the inhibition of human B cellproliferation induced by soluble gp39 and IL-4 when cells are culturedin the presence of anti-human gp39 mAbs 4D9-8, 4D9-9, 24-31, 2443, 89-76or 89-79.

FIG. 13 is a histogram representing the results of a proliferation assayof TT specific T cells from TT immunized hu-PBL-scid mice performed inthe absence of inhibitor (none) or in the presence of anti-human gp39antibody 24-31 or 89-76, or CD40Ig.

DETAILED DESCRIPTION OF THE INVENTION

This invention features methods for inducing antigen-specific T celltolerance. The methods involve contacting a T cell with 1) a cell whichpresents antigen to the T cell and has a ligand on the cell surface thatinteracts with a receptor on the surface of the T cell which mediatescontact dependent helper effector functions, and 2) an antagonist of thereceptor on the T cell which inhibits interaction of the receptor an theligand. As defined herein, a molecule or receptor which mediates contactdependent helper effector functions is one which is expressed on a Thcell and interacts with a ligand on an effector cell (e.g., a B cell),wherein the interaction of the receptor with its ligand is necessary forgeneration of an effector cell response (e.g., B cell activation). Inaddition to being involved in effector cell responses, it has now beenfound that such a molecule is involved in the response of the T cell toantigen.

A preferred molecule on a T cell which mediates contact-dependent helpereffector function is gp39. Accordingly, in preferred embodiments, themethods of the invention involve contacting a T cell with a cell whichpresents antigen and a gp39 antagonist. Accordingly, the cell used topresent antigen is one which interacts with gp39 on the surface of a Tcell to activate the T cell (i.e. deliver the necessary signals for Tcell activation to the T cell). For example, the cell can be a B cellwhich expresses CD40 and presents antigen to the T cell. By inhibitingan interaction between a gp39 ligand on the cell presenting antigen withgp39 on the T cell, the T cell is not activated by the presented antigenbut rather becomes tolerized to the antigen.

The methods of the invention can be used to induce T cell tolerance toan antigen in vivo. For example, a cell which presents antigen to a Tcell can be administered to a subject in conjunction with an antagonistof a receptor expressed on the T cell which mediates contact dependenthelper effector function (e.g. a gp39 antagonist). The methods of theinvention can further be used to tolerize a T cell to an antigen invitro by contacting the T cell in vitro with a cell which presentsantigen to the T cell together with an antagonist of a receptorexpressed on the T cell which mediates contact dependent helper effectorfunction (e.g. a gp39 antagonist). T cells tolerized in vitro can thenbe administered to a subject. The methods of the invention can be usedto tolerize T cells in a subject to a specific antigen, or totransplanted cells, such as allogeneic bone marrow (e.g., in bone marrowtransplantation). The methods of the invention are also useful forinhibiting graft versus host disease in bone marrow transplantation.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. gp39 Antagonists

According to the methods of the invention, a gp39 antagonist iscontacted with a T cell (e.g. administered to a subject) to interferewith the interaction of gp39 on a T cell with a gp39 ligand on anantigen presenting cell, such as a B cell. A gp39 antagonist is definedas a molecule which interferes with this interaction. The gp39antagonist can be an antibody directed against gp39 (e.g., a monoclonalantibody against gp39), a fragment or derivative of an antibody directedagainst gp39 (e.g., Fab or F(ab)'2 fragments, chimeric antibodies orhumanized antibodies), soluble forms of a gp39 ligand (e.g., solubleCD40), soluble forms of a fusion protein of a gp39 ligand (e.g., solubleCD40Ig), or pharmaceutical agents which disrupt or interfere with thegp39-CD40 interaction.

A. Antibodies

A mammal, (e.g., a mouse, hamster, or rabbit) can be immunized with animmunogenic form of gp39 protein or protein fragment (e.g., peptidefragment) which elicits an antibody response in the mammal. A cell whichexpresses gp39 on its surface can also be used as the immunogen.Alternative immunogens include purified gp39 protein or proteinfragments. gp39 can be purified from a gp39-expressing cell by standardpurification techniques; gp39 cDNA (Armitage et al., Nature, 357:80-82(1992); Lederman et al., J. Exp. Med., 175:1091-1101 (1992); Hollenbaughet al., EMBO J., 11:4313-4319(1992)) can be expressed in a host cell,e.g., bacteria or a mammalian cell line, and gp39 protein purified fromthe cell culture by standard techniques. gp39 peptides can besynthesized based upon the amino acid sequence of gp39 (disclosed inArmitage et al., Nature, 357:80-82 (1992); Lederman et al., J. Exp.Med., 175:1091-1101 (1992); Hollenbaugh et al., EMBO J., 11:4313-4319(1992)) using known techniques (e.g. F-moc or T-boc chemical synthesis).Techniques for conferring immunogenicity on a protein includeconjugation to carriers or other techniques well known in the art. Forexample, the protein can be administered in the presence of adjuvant.The progress of immunization can be monitored by detection of antibodytiters in plasma or serum. Standard ELISA or other immunoassay can beused with the immunogen as antigen to assess the levels of antibodies.

Following immunization, antisera can be obtained and, if desired,polyclonal antibodies isolated from the sera. To produce monoclonalantibodies, antibody producing cells (lymphocytes) can be harvested froman immunized animal and fused with myeloma cells by standard somaticcell fusion procedures thus immortalizing these cells and yieldinghybridoma cells. Such techniques are well known in the art. For example,the hybridoma technique originally developed by Kohler and Milstein(Nature (1975) 256:495-497) as well as other techniques such as thehuman B-cell hybridoma technique (Kozbar et al., Immunol. Today (1983)4:72), the EBV-hybridoma technique to produce human monoclonalantibodies (Cole et al. Monoclonal Antibodies in Cancer Therapy (1985)(Allen R. Bliss, Inc., pages 77-96), and screening of combinatorialantibody libraries (Huse et al., Science (1989) 246:1275). Hybridomacells can be screened immunochemically for production of antibodiesspecifically reactive with the protein or peptide and monoclonalantibodies isolated.

The term antibody as used herein is intended to include fragmentsthereof which are specifically reactive with a gp39 protein or peptidethereof or gp39 fusion protein. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as described above for whole antibodies. For example,F(ab')₂ fragments can be generated by treating antibody with pepsin. Theresulting F(ab')₂ fragment can be treated to reduce disulfide bridges toproduce Fab' fragments. The antibody of the present invention is furtherintended to include bispecific and chimeric molecules having ananti-gp39 portion.

When antibodies produced in non-human subjects are used therapeuticallyin humans, they are recognized to varying degrees as foreign and animmune response may be generated in the patient. One approach forminimizing or eliminating this problem, which is preferable to generalimmunosuppression, is to produce chimeric antibody derivatives, i.e.,antibody molecules that combine a non-human animal variable region and ahuman constant region. Chimeric antibody molecules can include, forexample, the antigen binding domain from an antibody of a mouse, rat, orother species, with human constant regions. A variety of approaches formaking chimeric antibodies have been described and can be used to makechimeric antibodies containing the immnunoglobulin variable region whichrecognizes gp39. See, for example, Morrison et al., Proc. Natl. Acad.Sci. U.S.A. 81:6851 (1985); Takeda et al., Nature 314:452 (1985),Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No.4,816,397; Tanaguchi et al., European Patent Publication EP171496;European Patent Publication 0173494, United Kingdom Patent GB 2177096B.It is expected that such chimeric antibodies would be less immunogenicin a human subject than the corresponding non-chimeric antibody.

For human therapeutic purposes the monoclonal or chimeric antibodiesspecifically reactive with a gp39 protein or peptide can be furtherhumanized by producing human variable region chimeras, in which parts ofthe variable regions, especially the conserved framework regions of theantigen-binding domain, are of human origin and only the hypervariableregions are of non-human origin. Such altered immunoglobulin moleculesmay be made by any of several techniques known in the art, (e.g., Tenget al., Proc. Natl. Acad. Sci. U.S.A., 80:7308-7312 (1983); Kozbor etal., Immunology Today, 4:7279 (1983); Olsson et al., Meth. Enzymol.,92:3-16 (1982)), and are preferably made according to the teachings ofPCT Publication W092/06193 or EP 0239400. Humanized antibodies can becommercially produced by, for example, Scotgen Limited, 2 Holly Road,Twickenham, Middlesex, Great Britain.

Another method of generating specific antibodies, or antibody fragments,reactive against a gp39 protein or peptide is to screen expressionlibraries encoding immunoglobulin genes, or portions thereof, expressedin bacteria with a gp39 protein or peptide. For example, complete Fabfragments, VH regions and FV regions can be expressed in bacteria usingphage expression libraries. See for example Ward et al., Nature, 341:544-546: (1989); Huse et al., Science, 246: 1275-1281 (1989); andMcCafferty et al., Nature, 348: 552-554 (1990). Screening such librarieswith, for example, a gp39 peptide can identify imunoglobin fragmentsreactive with gp39. Alternatively, the SCID-hu mouse (available fromGenpharm) can be used to produce antibodies, or fragments thereof.

Methodologies for producing monoclonal antibodies directed against gp39,including human gp39 and mouse gp39, and suitable monoclonal antibodiesfor use in the methods of the invention, are described in further detailin Example 6. Particularly preferred anti-human gp39 antibodies of theinvention are mAbs 24-31 and 89-76, produced respectively by hybridomas24-31 and 89-76. The 89-76 and 24-31 hybridomas, producing the 89-76 and24-31 antibodies, respectively, were deposited under the provisions ofthe Budapest Treaty with the American Type Culture Collection, ParklawnDrive, Rockville, Md., on Sep. 2, 1994. The 89-76 hybridoma was assignedATCC Accession Number HB1 1712 and the 24-31 hybridoma was assigned ATCCAccession Number HB 11713.

Recombinant anti-gp39 antibodies, such as chimeric and humanizedantibodies, can be produced by manipulating nucleic acid (e.g., DNA)encoding an anti-gp39 antibody according to standard recombinant DNAtechniques. Accordingly, another aspect of this invention pertains toisolated nucleic acid molecules encoding immunoglobulin heavy or lightchains, or portions thereof, reactive with gp39, particularly humangp39. The immunoglobulin-encoding nucleic acid can encode animmunoglobulin light or heavy chain variable region, with or without alinked heavy or light chain constant region (or portion thereof). Suchnucleic acid can be isolated from a cell (e.g., hybridoma) producing ananti-human gp39 mAb by standard techniques. For example, nucleic acidencoding the 24-31 or 89-76 mAb can be isolated from the 24-31 or 89-76hybridoma, respectively, by cDNA library screening, PCR amplification orother standard technique. Following isolation of, and possible furthermanipulation of, Moreover, nucleic acid encoding an anti-human gp39 mAbcan be incorporated into an expression vector and introduced into a hostcell to facilitate expression and production of recombinant forms ofanti-human gp39 antibodies.

B. Soluble Ligands for gp39

Other gp39 antagonists which can be used to induce T cell tolerance aresoluble forms of a gp39 ligand. A monovalent soluble ligand of gp39,such as soluble CD40 can bind gp39, thereby inhibiting the interactionof gp39 with CD40 on B cells. The term "soluble" indicates that theligand is not permanently associated with a cell membrane. A solublegp39 ligand can be prepared by chemical synthesis, or, preferably byrecombinant DNA techniques, for example by expressing only theextracellular domain (absent the transmembrane and cytoplasmic domains)of the ligand. A preferred soluble gp39 ligand is soluble CD40.Alternatively, a soluble gp39 ligand can be in the form of a fusionprotein. Such a fusion protein comprises at least a portion of the gp39ligand attached to a second molecule. For example, CD40 can be expressedas a fusion protein with immunoglobulin (i.e., a CD40Ig fusion protein).In one embodiment, a fusion protein is produced comprising amino acidresidues of an extracellular domain portion of the CD40 molecule joinedto amino acid residues of a sequence corresponding to the hinge, CH2 andCH3 regions of an immunoglobulin heavy chain, e.g., Cγ1, to form aCD40Ig fusion protein (see e.g., Linsley et al. (1991) J. Exp. Med.1783:721-730; Capon et al. (1989) Nature 337, 525-531; and Capon U.S.Pat. No. 5,116,964). The fusion protein can be produced by chemicalsynthesis, or, preferably by recombinant DNA techniques based on thecDNA of CD40 (Stamenkovic et al., EMBO J., 8:1403-1410 (1989)).

II. Cells for Induction of Antigen-Specific Tolerance

The current invention is based, at least in part, on the discovery thatpresentation of an antigen to a T cell by a cell which both presentsantigen and interacts with gp39 results in antigen-specific T celltolerance when the antigen is presented to the T cell in the presence ofa gp39 antagonist. Cells which are capable of inducing T cell toleranceby this mechanism include those which present antigen to a T cell andrequire an interaction between a gp39 ligand on the cell and gp39 on theT cell to deliver the necessary signals for T cell activation to the Tcell. Inhibition of this interaction prevents T cell activation by thepresented antigen and, rather, induces antigen-specific tolerance in theT cell. Interference with activation of the T cell via gp39 may preventthe induction of costimulatory molecules on the antigen presenting cell(e.g., B7 family molecules on an antigen presenting cell such as a Bcell) so that the antigen presenting cell delivers only an antigenicsignal in the absence of a costimulatory signal, thus inducingtolerance.

Accordingly, in the methods of the invention, a cell which presentsantigen is administered to a recipient subject. The phrase "cell whichpresents antigen" and "antigen presenting cell" are used interchangeablyherein and are intended to encompass cells which can present antigen toT cells of the recipient and includes B lymphocytes, "professional"antigen presenting cells (e.g., monocytes, dendritic cells, Langerhancells) and other cells which present antigen to immune cells (e.g.,keratinocytes, endothelial cells, astrocytes, fibroblasts,oligodendrocytes). Furthermore, it is preferable that the antigenpresenting cell have a reduced capacity to stimulate a costimulatorysignal in recipient T cells. For example, the antigen presenting cellmay lack expression of or express only low levels of costimulatorymolecules such as the B7 family of proteins (e.g., B7-1 and B7-2).Expression of costimulatory molecules on potential antigen presentingcells to be used in the method of the invention can be assessed bystandard techniques, for example by flow cytometry using antibodiesdirected against costimulatory molecules.

Preferred antigen presenting cells for inducing T cell tolerance arelymphoid cells, for example peripheral blood lymphocytes or spleniccells. Preferred lymphoid cells for inducing T cell tolerance are Bcells. B cells can be purified from a mixed population of cells (e.g.,other cell types in peripheral blood or spleen) by standard cellseparation techniques. For example, adherent cells can be removed byculturing spleen cells on plastic dishes and recovering the non-adherentcell population. T cells can be removed from a mixed population of cellsby treatment with an anti-T cell antibody (e.g., anti-Thy1.1 and/oranti-Thy1.2) and complement. In one embodiment, resting lymphoid cells,preferably resting B cells, are used as the antigen presenting cells.Resting lymphoid cells, such as resting B cells, can be isolated bytechniques known in the art, for example based upon their small size anddensity. Resting lymphoid cells can be isolated for example bycounterflow centrifugal elutriation as described in Tony, H-P. andParker, D. C. (1985) J. Exp. Med 161:223-241. Using counterflowcentrifugal elutriation, a small, resting lymphoid cell populationdepleted of cells which can activate T cell responses can be obtained bycollecting a fraction(s) at 14-19 ml/min., preferably 19 ml/min. (at3,200 rpm). Alternatively, small, resting lymphocytes (e.g., B cells)can be isolated by discontinuous density gradient centrifugation, forexample using a Ficoll or Percoll gradient, and a layer containingsmall, resting lymphocytes can be obtained after centrifugation. Smallresting B cells can also be distinguished from activated B cells byassaying for expression of costimulatory molecules, such as B7-1 and/orB7-2, on the surface of activated B cells by standard techniques (e.g.,immunofluorescence).

The antigen presenting cell, such as a B cell, can be contacted with anantigen (e.g., a soluble protein) prior to contact with the T cell(e.g., prior to administration to a subject) and the cell used topresent the antigen to the T cell in the presence of a gp39 antagonistto induce specific T cell tolerance to the antigen (see Example 1).Alternatively, tolerance to alloantigens can be induced by using anallogeneic cell as the antigen presenting cell (see Examples 2 and 3).The allogeneic cell presents antigenic fragments of allogeneic proteinsto T cells. In one embodiment, the allogeneic cell is an allogeneiclymphoid cell, such as an allogeneic B cell. Alternatively, a subjectcan be tolerized with cells in peripheral blood (e.g., peripheral bloodlymphocytes), splenic cells or bone marrow cells. In the case of bonemarrow transplantation, the donor bone marrow cells themselves serve asthe antigen presenting cells contacted with the T cells (e.g.,administered to a subject). Accordingly, allogeneic bone marrow can beadministered in conjunction with a gp39 antagonist to induce toleranceto the bone marrow in the recipient and to prevent graft versus hostdisease (see Examples 4 and 5).

III. Administration of Cells and gp39 Antagonists

Antigen-specific T cell tolerance can be induced according to theinvention by administration of a gp39 antagonist to a subject inconjunction with a cell which presents antigen to a T cell and expressesa ligand which interacts with gp39 on the T cell. In a preferredembodiment, the antigen presenting cell and the gp39 antagonist areadministered simultaneously or contemporaneously. Alternatively, thegp39 antagonist can be administered prior to administering the cells,for example when the antagonist is an antibody with a long half-life. Ina case where the cells to be administered are bone marrow cells, whereininhibition of graft-versus-host disease is desired, the donor T cells inthe bone marrow can be tolerized before transfer to the recipient hostby incubating the donor bone marrow with B cells from the host and agp39 antagonist in vitro. gp39 treatment can be continued in vivo duringand after bone marrow transfer if necessary. In subjects who are toreceive a bone marrow or other organ transplant, allogeneic tolerance tothe transplant can be induced in the subject by treatment with a regimenwhich induces allogeneic tolerance prior to transfer of the organ orbone marrow cells. This pre-treatment regimen can involve administeringto the subject cells from the donor together with a gp39 antagonist. Thecells from the donor can be, for example, B cells, whole peripheralblood or some fraction thereof (e.g., peripheral blood lymphocytes orsmall resting lymphocytes or B cells).

Administration of a single dose of antigen presenting cells (incombination with the antagonist) has been found to be sufficient forinduction of T cell tolerance (see the Examples). The number of antigenpresenting cells administered may vary depending upon the type of cellused, the type of tissue or organ graft, the weight of the recipient,the general condition of the recipient and other variables known to theskilled artisan. An appropriate number of cells for use in the method ofthe invention can be determined by one of ordinary skill in the art byconventional methods (for example using assays described in theExamples). Cells are administered in a form and by a route which issuitable for induction of tolerance in the recipient. Cells can beadministered in a physiologically acceptable solution, such as abuffered saline solution or similar vehicle. Cells are preferablyadministered intravenously.

An antagonist of the invention is administered to subjects in abiologically compatible form suitable for pharmaceutical administrationin vivo to induce T cell tolerance. By "biologically compatible formsuitable for administration in vivo" is meant a form of the antagonistto be administered in which any toxic effects are outweighed by thetherapeutic effects of the protein. The term subject is intended toinclude living organisms in which an immune response can be elicited,e.g., mammals. Examples of subjects include humans, dogs, cats, mice,rats, and transgenic species thereof. A gp39 antagonist can beadministered in any pharmacological form, optionally in apharmaceutically acceptable carrier. Administration of a therapeuticallyactive amount of the antagonist is defined as an amount effective, atdosages and for periods of time necessay to achieve the desired result.For example, a therapeutically active amount of an antagonist of gp39may vary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antagonist to elicit adesired response in the individual. Dosage regima may be adjusted toprovide the optimum therapeutic response. For example, several divideddoses may be administered daily or the dose may be proportionallyreduced as indicated by the exigencies of the therapeutic situation.

The active compound (e.g., antagonist) may be administered in aconvenient manner such as by injection (subcutaneous, intravenous,etc.), oral administration, inhalation, transdermal application, orrectal administration. Depending on the route of administration, theactive compound may be coated in a material to protect the compound fromthe action of enzymes, acids and other natural conditions which mayinactivate the compound. A preferred route of administration is byintravenous injection.

To administer an antagonist of gp39 by other than parenteraladministration, it may be necessary to coat the antagonist with, orco-administer the antagonist with, a material to prevent itsinactivation. For example, an antagonist can be administered to anindividual in an appropriate carrier or diluent, co-administered withenzyme inhibitors or in an appropriate carrier such as liposomes.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Enzyme inhibitors include pancreatic trypsin inhibitor,diisopropylfluorophosphate (DEP) and trasylol. Liposomes includewater-in-oil-in-water emulsions as well as conventional liposomes(Strejan et al., (1984) J. Neuroimmunol 7:27).

The active compound may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, asorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating activecompound (e.g., an antagonist of gp39) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient (e.g., antagonist) plusany additional desired ingredient from a previously sterile-filteredsolution thereof.

When the active compound is suitably protected, as described above, theprotein may be orally administered, for example, with an inert diluentor an assimilable edible carrier. As used herein "pharmaceuticallyacceptable carrier" includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the therapeutic compositions iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

In addition to tolerization of T cell in vivo, the invention encompassestolerization of T cells in vitro, e.g., by contact with an antigenpresenting cell in the presence of a gp39 antagonist. For example, Tcells can be obtained from a subject, tolerized in vitro by culture withthe antigen presenting cells and the antagonist and then readministeredto the subject.

IV. Uses of the Method of the Invention

The methods of the invention can be applied to induce T cell toleranceto a variety of antigens. For example, T cell tolerance can be inducedto a soluble antigen (e.g., a soluble protein), as described inExample 1. T cells can be tolerized to antigens involved in autoimmunediseases or disorders associated with abnormal immune responses. Forexample, in one embodiment, the antigen is an autoantigen. In anotherembodiment, the antigen is an allergan. Alternatively, T cells can betolerized to antigens expressed on foreign cells (as described inExamples 2 to 5). Accordingly, in yet other embodiments, the antigen isan alloantigen or xenoantigen. Induction of T cell tolerance toalloantigens and xenoantigens is of particular use in transplantation,for example to inhibit rejection by a transplant recipient of a donorgraft, e.g. a tissue or organ graft or bone marrow transplant.Additionally, tolerization of donor T cells within a bone marrow graftis useful for inhibiting graft versus host disease (see Example 5).

The invention is further illustrated by the following exampes whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference.

EXAMPLE 1 Induction of Antigen-Specific Tolerance

Methods

Mice were immunized with KLH-pulsed splenic B lymphocytes for 5 days.During the primary immunization animals were either untreated or treatedwith an anti-gp39 antibody MR1. Five days after the initialimmunization, mice were given a local (food pad) challenge with KLH incomplete Freund's adjuvant (CFA). Mice were sacrificed 5 days later, thedraining lymph nodes removed and the T cell proliferative response toKLH was subsequently assayed in vitro.

Results

Animals immunized with activated B lymphocytes pulsed with antigen andsubsequently challenged with the same antigen mount significant immuneresponses to the immunizing antigen. The response measured byproliferation of antigen-specific T lymphocytes is shown in FIG. 1.Treatment of animals with an anti-gp39 antibody during the primaryimmunization results in unresponsiveness of antigen-specific Tlymphocytes upon in vitro antigen challenge. T lymphocytes obtained fromlymph nodes from animals treated with an anti-gp39 antibody showdecreased proliferative capacity when compared to T lymphocytes obtainedfrom lymph nodes of untreated animals.

EXAMPLE 2 Induction of T Cell Tolerance to Allogeneic Cells

Methods

Balb/c (H-2^(d)) mice were immunized with allogeneic splenic B cellsfrom DBA/2 (H-₂ ^(b)) mice. Animals were treated with an anti-gp39antibody, MR1, or untreated for 5 days after immunization. On day 6, theanimals were sacrificed and spleens removed. Spleen cells from anti-gp39antibody or untreated animals were subsequently cultured in vitro witheither no stimulus or with irradiated DBA/2 spleen cells. Theproliferative response to this secondary allogeneic stimulation wasmeasured on day 3 after initiation of culture.

Results

T lymphocytes from animals immunized with allogeneic splenic B cellsmount strong proliferative responses when challenged 5 days later withthe same cells in vitro (FIG. 2). However, T lymphocytes from miceimmunized with allogeneic splenic B cells and treated with an anti-gp39antibody have a decreased proliferative capacity when subsequentlychallenged ion vitro. Anti-gp39 antibody treated mice exhibitapproximately 50% decrease in responsiveness to challenge compared tocontrol untreated mice.

EXAMPLE 3 Anti-gp39 Treatment Interferes with the Generation of CTLResponses to Allogeneic B Cells

In this example, the role of gp39 in the generation of cytotoxic T cells(CTL) was investigated. To assess the in vivo function of gp39 in thedevelopment of CTL, the effects of anti-gp39 treatment on the generationof allospecific CTL by immunization with allogeneic B cells wasexamined. Effects of anti-gp39 treatment of both primary and secondaryCTL responses were determined.

Primary CTL Responses

To test whether anti-gp39 can prevent allogeneic B cells from elicitingallospecific CTL responses in vivo, allogeneic B cells (T-depletedspleen cells) were administered to recipient mice with or withoutanti-gp39. Balb/c mice (female, 6-8 weeks old, Jackson Laboratories, BarHarbor, Me.) were immunized with C57BL/6 (female, 6-8 weeks old, JacksonLaboratories, Bar Harbor, Me.) spleen cells (30-50×10⁶) depleted of Tcells by anti-Thy 1.2 (ascites prepared from ATCC clone HO13.4) andrabbit complement treatment. These recipient mice were then untreated ortreated with anti-gp39 for 5 days (250 mg/recipient on days 0, 2 and 4).On day five, spleens were removed and CTL responses were measured usinga 4 hour chromium release assay. Spleen cells from unprimed Balb/c micewere used as negative control effector cells. Target cells used were Efemale K1 (H-2^(b), T cell lymphoma derived from C57BL/6 strain) andP815 (H-₂ ^(d), mastocytoma, derived from DBA/2J strain). ⁵¹ Cr-labelledtarget cells were washed and plated at 1×10⁴ cells per well in 96 wellplates with effector cells in effector:target (E:T) ratios of 100:1,20:1 and 4:1. The plates were briefly centrifuged and then incubated at37° C. in 5% CO₂ for 4 hours. The plates were once more centrifuged and100 ml of cell-free supernatant was collected from each well for gammacounting (LKB Clinigamma, Wallace Inc., Gaithersburg, Md.). Percentspecific lysis is defined as (a-b)/c, where a=cpm released by targetcells incubated with effector cells, b=cpm released by target cellsincubated with media only (spontaneous release) and c=freeze-thawreleasable cpm from target cells (approximately 80% of total cpmincorporated). P815 targets were not lysed by any of the cell samplestested. Results for E female K1 targets are illustrated in FIG. 3A,wherein the groups shown are untreated mice (▪), anti-gp39 treated mice(Δ) and spleen cells from unprimed Balb/c mice (; used as negativecontrol effector cells). The results demonstrate that mice that receivedanti-gp39 and allogeneic cells did not generate a primary CTL responsein vivo in response to allogeneic B cells. In contrast, in the absenceof anti-gp39, the allogeneic B cells sensitized the recipient toalloantigen and induced a substantial CTL response.

To determine if anti-gp39 could only amplify the tolerogenic effect ofnon-activated, splenic B cells, LPS-activated B cells were used to primeCTL in the presence or absence of anti-gp39. B cells from C57BL/6 micewere prepared as described above and cultured for 2 days in the presenceor absence of lipopolysaccharide (LPS; 50 mg/ml; Sigma Diagnostics, StLouis, Mo.). Cells were then harvested and washed thoroughly andinjected i.p. (30-50×10⁶) into Balb/c recipient mice. Recipient micewere either untreated or treated with anti-gp39 on days 0, 2 and 4 asdescribed above. On day 5, spleens were removed and CTL responses weredetermined as described above. The results of two independentexperiments are shown in FIG. 3B (top and bottom panels). Groupsrepresented are LPS blasts in vivo without treatment (>), LPS blasts invivo with anti-gp39 treatment (), resting B cells in vivo withouttreatment (◯) and resting B cells in vivo with anti-gp39 treatment (□).The results indicate that, although not complete, anti-gp39 treatmentalso reduced the primary CTL response to allogeneic LPS blasts.

Secondary CTL Responses

To examine the impact of anti-gp39 and allogeneic B cell administrationon CTL formation, spleen cells from treated and untreated mice werestimulated in vitro and the scope of CTL responses investigated. Balb/cmice were primed in vivo with C57BL/6-derived B cells as describedabove. Spleen cells from the anti-gp39 and control hamster Ig(HIg)-treated animals were removed and 5×10⁶ cells/ml were cultured for5 days ("responders") with various mitomycin C treated (2.5 mg/ml at 37°C., Sigma Diagnostics, St. Louis, Mo.) strains of splenic B cells(prepared by anti-Thy1.2+complement treatment) at 5×10⁶ stimulators/ml.Stimulator groups were C57BL/6 (H-₂ ^(b)) and Balb/c (H-₂ ^(d)) whichindicate the secondary allogeneic response and the primary syngeneicresponse, respectively. Stimulator and responder cells were cultured infreshly prepared sensitization RPMI-1640 medium (Bio Whittaker,Walkersville, Md.), 1×10⁵ M 2-mercaptoethanol (Bio-Rad Laboratories,Hercules, Calif.), 2 mM L-glutamine (Sigma Diagnostics, St. Louis, Mo.),500 U/ml penecillin and 5000 U/ml streptomycin (Sigma Diagnostics, St.Louis, Mo.). After 5 days, the responder cells were harvested and deadcells were removed by centrifugation on a density gradient. Theresulting live cells were used as effectors in a CTL assay as describedabove. Targets included E female K1 (H-₂ ^(b), T cell lymphoma derivedfrom C57BL/6 strain) and P815 (H-₂ ^(d), mastocytoma, derived fromDBA/2J strain). The results are illustrated in FIG. 4A. Allogeneicstimulated effector groups shown are: HIg treated recipients (), naiveBalb/c (>) and anti-gp39 treated recipients (▪). Corresponding syngeneicresponse (Balb/c cells stimulated with Balb/c cells) are indicated byopen symbols. As anticipated, in vivo immunization of mice withallogeneic T-depleted spleen cells (H-₂ ^(b)) in the absence ofanti-gp39 treatment resulted in a robust secondary CTL response invitro. The heightened secondary anti-H-2^(b) CTL response was notobserved if mice were given anti-gp39 in vivo.

To determine the specificity of the inhibition of CTL responses byanti-gp39 treatment, spleen cell cultures as described above wereanalyzed for anti-H-2^(k) allogeneic responses (third party allogeneicresponses) using B10BR (H-₂ ^(k)) splenic B cells as the stimulators andSL8 (H-₂ ^(k), spontaneous T cell lymphoma derived from AKR strain) asthe targets in the CTL assay. The results are shown in FIG. 4B. Groupsshown are naive Balb/c (◯) and anti-gp39 treated recipients (▪). Theresults demonstrate that the inhibition of anti-H2^(b) -specific CTLresponses by anti-gp39 treatement was allospecific since administrationof H-2^(b) spleen cells and anti-gp39 did not alter the in vitro CTLresponse to a bystander alloantigen (H-₂ ^(k)).

Taken together, these data show that anti-gp39 interferes with thegeneration of allospecific CTL responses (both primary and secondary).In the presence of anti-gp39, CTL precursors specific to the relevantalloantigen are still present since in the in vitro secondary cultureone can demonstrate some anti-H-2^(b) CTL activity. It can be concludedthat anti-gp39 treatment reduced the secondary in vitro response byblocking CTL priming or reducing the frequency of primed allospecificCTL. The use of resting B cells is not imperative to demonstrate thisunresponsiveness since the allogeneic CTL response induced by LPS blastswas also impaired by anti-gp39.

EXAMPLE 4 Transfer of Allogeneic Murine Bone Marrow Induces StableChimeras when Recipients are Treated with an Antibody to gp39

The treatment for many hematological disorders, that cannot be curedwith conventional chemotherapeutic methods, involves the transfer ofallogeneic bone marrow. This aggressive therapy involves administrationof high, myeloablative doses of chemotherapy coupled with the perfusionof allogeneic bone marrow allowing the eradication of clonogenic tumorcells.

It has already been shown that thymic irradiation, deleting thymic Tcells, increases the incidence of stable chimeras when monoclonalantibodies (mAbs) to T cell, including anti-CD4 mAbs were used. Thisantibody regime led to T cell deletion and thus donor specifictolerance, which could be indicated by tolerance to donor skin grafts.Failure to achieve permanent allogeneic engraftment after 300 rad WBI,in vivo anti-T cell mAb treatment, and administration of allogeneic bonemarrow may have resulted from the unaffected T cells within the thymus.Use of anti-CD4 and anti-CD8 mAbs can lead to effective depletion ofperipheral blood and splenic T cells, but the thymic T cells are simplycoated with mAbs but not deleted. Thus thymic irradiation is performedon the mice.

Methods

A murine model for allogeneic bone marrow transfer (BMT) of F1 intoparent was used so as to avoid graft-versus-host disease from beinginduced. We used the F1 strain CB6 which are (C57BL/6×Balb/c)F1resulting the overall MHC haplotype of H-2^(d/b). Bone marrow wasremoved and i.v. injected into the irradiated BALB/c parent with orwithout an anti-gp39 antibody, MR1. Whole body irradiation (WBI) levelswere varied so as to determine the best level of chimerism and tocompare anti-gp39 antibody treated and untreated animals with eachirradiation level. Chimerism was determined by flow cytometric analysisof peripheral blood lymphocytes of the H-2^(b) MHC haplotype presentwithin the H-₂ ^(d) (BALB/c) mouse. It has previously been shown thatthis combination of mice is suitable and chimeras can be obtained atlevels of 500 and 600 rads WBI.

Results

Chimerism was detected by identification of C57BL/6 derived cells (H-₂^(b)) by flow cytometry. Chimerism was found to develop in miceirradiated with 400, 500 and 600 whole body irradiation within 14 daysonly if treated with anti-gp39 antibody from the start of the study.Mice that received no antibody treatment rejected the cells at alllevels of irradiation except when given 600 whole body irradiation,whereby they accepted the bone marrow to the same extent as the antibodytreated group at 400 rads (Table 1). It was found that the level ofchimerism after 6 weeks of therapy at 400, 500 and 600 rads withanti-gp39 antibody therapy to be 70.7+5.74, 94.1 and 84.4+8.56respectively, while at 600 rads with no treatment they it was found tobe 85.7+5.9 chimeric (Table 2). Phenotyping the cells at this pointindicated that T cells, B cells and macrophages were all chimerised andthe the same extent for 400 rads when treated compared to the 600 radsuntreated group (Table 2). It also appeared that anti-gp39 antibodytreatment did not significantly change the total percent population ofany of the lymphoid cells within the periphery. When treatment of theanimals with anti-gp39 antibody was terminated the animals were found tobe stably chimeric for up to 8 weeks post transplant.

                  TABLE I    ______________________________________    Whole      No. of chimeric animals                              No. of chimeric animals    body radiation               treated with   with no anti-gp39    level (rads)               anti-gp39 treatment                              treatment    ______________________________________    0          0(5)           0(5)    200        0(5)           0(5)    400        9(9)           0(9)    450        3(5)           0(5)    500        4(5)           2(5)    600        7(9)           7(9)    ______________________________________     Levels of whole body irradiation resulting in chimerism. Numbers in     parenthesis indicate the total number of animal tested at each level.

                  TABLE 2    ______________________________________         Treat-            B cells T cells    Rads.         ment    II-2K.sup.b+                           II-2K.sup.b+                                   II-2K.sup.b+                                           Mac. II-2K.sup.b+    ______________________________________    400  +       70.7 ± 5.74                           89 ± 2.6                                   45.9 ± 13.3                                           82.3 ± 3.3    500  +       94.1      100     89      100    600  +       84.4 ± 8.56                           99.3 ± 0.94                                   72.3 ± 15.9                                           91.1 ± 8.3    600  -       85.7 ± 5.9                           97.3 ± 1.77                                   67.1 ± 10.3                                           91 ± 9    ______________________________________     Percent of B cell, T cell and macrophages (Mac.) positive for H2Kb+     standard error.

EXAMPLE 5 Allogeneic Bone Marrow Transplantation Combined with Treatmentof the Recipient with Anti-gp39 Inhibits Acute and Chronic Graft VersusHost Disease

The following methodology was used in this example:

Mice: DBA/2 (H-2^(d)), C57BL/6 (H-2^(b)) and B6D2F₁ ((C57BL/6 (H-₂^(d))×DBA/2) F₁ hybrid) mice were obtained from the NCI laboratories(Bethesda, Md.) and maintained in a viral free environment in the Animalfacility at Dartmouth Medical School. All the mice used in this studywere female, and aged 6 to 8 weeks old.

Induction of chronic GVHD: Chronic GVHD was induced by the i.v.injection of parental (DBA/2) spleen cells into non-irradiated(C57BL/6×DBA/2) F₁ hybrid recipients (Fast, L. D. (1990) J. Immunol.144:4177). Parental mice were anesthetized and killed by cervicaldislocation in preparation for removal of the spleen. Dissociated spleencells were washed and resuspended in RPMI 1640 medium (Whittaker,Waldersville, Md.) for i.v. injection into the F₁ recipients.

Induction of acute GVHD: Acute GVHD was induced by the i.v. injection ofparental C57BL/6 spleen cells into non-irradiated (C57BL/6×DBA/2) F₁hybrid recipients. Cells were prepared for transfer as for the inductionof chronic GVHD.

Antibodies: Anti-gp39:MR1 was produced in ascites and purified by ionexchange HPLC, as previously described (Foy, T. M. et al. (1993) J. Exp.Med. 178:1567-1575; Noelle, R. J. et al. (1992) Proc. Natl. Acad. Sci.USA 89:6550). Polyclonal anti-isotype antibodies: All anti-IgG₁ and IgAantibodies and standard controls were obtained from SouthernBiotechnology Associates, Inc., (Birmingham Ala.). Anti-IgE antibodies:All anti-IgE antibodies (BIE3 and Biotin AM95) and standards (A3B1) usedin the IgE specific ELIZA were a kind gift from Dr. T. Waldschmidt, IA.Anti-MHC halpotype antibodies: H-2K^(b) FITC conjugated antibody andH-2D^(d) Biotin conjugated antibody were obtained from PharMingen (SanDiego, Calif.)

Cell lines: Cell lines used included P815 (H-2^(d)) and LB27.4 (H-₂^(bxd)), which were obtained from the American Type Culture Collection.The cell line c 1.18.5 (H-2b) was obtained as a kind gift from Dr.William R. Green.

Polyclonal Ig production in vitro: Spleens from control and cGVHD micewere removed and single cell suspensions prepared. Cells were treatedwith Tris-buffered ammonium chloride to remove erythrocytes and totalwhite blood cell counts determined by visual hemocytometer counting.Cells were incubated (5×10⁶) in 1 ml of complete (c)RPMI-1640 medium(supplemented with 10% fetal serum (Hyclone, Logan Utah, 25 mM HEPES, 2mM L-glutamine, 5000 U/ml penicillin and 5000 mg/ml streptomycin) for 3days at 37° C., 5% CO₂. Culture supernatants were collected by pelletingthe cells and Ig was quantified by an isotype-specific ELISA assay.

Isotype-specific and antigen-specific ELISAs: ELISA for the detection ofIgG₁ and IgA: Goat anti-mouse IgG₁ or IgA (10μg/ml; SouthernBiotechnology Associates, Inc., Birmingham Ala.) in PBS was absorbedonto wells of a 96-well polyvinyl microtitre plate for 1 hour at 37° C.then overnight at 4° C. Plates were washed and blocked with PBScontaining 1% FCS for 1 hour at 37° C. Plates were washed again and theappropriate dilutions of supernatants and standard controls (IgG₁ andIgA, Southern Biotechnology Associates, Inc., Birmingham Ala.) wereadded for 2 hours at 37° C. After this time, the plates were washed 3times and alkaline-phosphatase conjugated goat anti-mouse IgG₁ or IgA,(1/500 dilutions) (Southern Biotechnology Associates, Inc., BirminghamAla.) were added for 2 hours at 37° C. Plates were thoroughly washed andphosphatase substrate (1 mg/ml; Sigma Diagnostics, St. Louis, Mo.) addedresulting in the appropriate colour change. Readings were determined byan ELISA reader (Dynatech Laboratories, Inc.) at an absorbance of 410nm. Concentrations of Ig were determined by comparison to theappropriate isotype standard curve and expressed as the mean+standarderror (n=3).

ELISA for the detection of IgE: Wells of 96-well polyvinyl microtitreplate were coated with an anti-mouse IgE capture antibody (B 1E3(2mg/ml)) overnight at 4° C. and then blocked with PBS containing 1% FCSfor 1 hour at 37° C. Plates were washed again and the appropriatedilutions of supernatants and standard controls (A3B 1 (IgE)) were addedfor 2 hours at 37° C. Plates were thoroughly washed and the EM95-Biotin(5mg/ml) was added to each well and incubated for 2 hours at 37° C.After this time, alkaline-phosphatase conjugated to streptavadin wasadded (1/500 dilution) for a further 2 hours and then washed thoroughlybefore the addition of phosphatase substrate (1 mg/ml; SigmaDiagnostics, St. Louis, Mo.) resulting in the appropriate colour change.Readings were determined by an ELISA reader (Dynatech Laboratories,Inc.) at an absorbance of 410 nm. Concentrations of Ig were determinedby comparison to standard curve and expressed as the mean+standard error(n=3).

ELISA for the detection of anti-DNA Ab: Calf thymus DNA (Sigma, St.Louis, Mo.) (5μg/ml) was dissolved in coupling buffer containing 0.1Msodium carbonate/sodium bicarbonate (pH 9.8). This was boiled for 10minutes and then incubated on ice for 3 minutes. The OD 260 of the DNAwas then determined and the concentration adjusted to obtain therequired 5 μg/ml DNA. 100,l was then added to the wells of a 96 wellpolyvinyl microtitre plate and incubated overnight at 4° C. The platewas then washed 3 times and blocked for 1 hour at 37° C. with PBScontaining 1% FCS and 0.02% sodium azide. The plate was again washed andserial dilutions of serum samples was then added (100 ml) and incubatedfor 2 hours at 37° C. The detection antibody, goat anti mouse IgG₁alkaline phosphatase was then added each well and incubated once againfor 2 hours at 37° C. Plates were thoroughly washed and phosphatasesubstrate (1 mg/ml; Sigma Diagnostics, St. Louis, Mo.) added resultingin the appropriate colour change. Readings were determined by an ELISAreader (Dynatech Laboratories, Inc.) at an absorbance of 410 nm.Antibody titres were compared to a positive sera samples and resultsexpressed in arbitrary units.

Flow Cytometric Analysis for the detection of donor derived cells.Spleens were removed from normal BDF₁, cGVHD mice treated with andwithout anti-gp39 and a single cell suspension was prepared. The cellswere layered onto Ficoll-Hypaque (4:1) and then contrifuged at 2000 rpmfor 20 minutes at room temperature. The resulting lymphocyte layer wasremoved and washed once with BSS containing 5% FCS. 1×10⁶ cells, pertube, were used for staining in a 50 μl final volume. 50 μl of rat serumwas added to each tube to prevent non specific binding of antibodies.Cells were stained with the (a) control antibodies: Rat Ig FITC (1:100final dilution) and PE-Streptavidin (1:500 final dilution) and (b) FITCH-2K^(b) and Biotin H-2D^(d) (both 1:100 final dilution). Cells wereincubated for 20 minutes on ice and then washed twice to remove anyunbound antibodies. Finally PE-Streptavidin (1:500 final dilution) wasadded to the appropriate tube to detect the biotin conjugated antibodyfor another 20 minutes on ice. Cells were again washed twice ready foranalysis on a Becton Dickinson FACScan. After positive gating viaforward and side scatter, 10,000 events were collected per sample fordetermination of percent cells positive for the relevant MHC haplotype.

Chromium release assay for assessment of CTL assay. ⁵¹ Cr-labelledtarget cells are washed and plated at 1×10⁴ per well in 96 well plateswith effector cells in E:T ratios of 100:1, 20:1, and 4:1. Target cellsused included P815 (H-2d), LB27.4 (H-2^(bxd)) and c118.5 (H-₂ ^(b)). Theplates are briefly centrifuged and then incubated at 37° C. in 5% CO₂for 4 hours. The plates were once more centrifuged and an aliquot ofcell-free supernatant was collected from each well for counting by agamma counter. Percent specific lysis is defined as (a-b)/c where a=cpmreleased by target cells incubated with effector cells, b=cpm releasedby target cells incubated with media only (spontaneous release), andc=freeze-thaw releasable cpm for target cells (approximately 80% oftotal cpm incorporated).

Secondary stimulation of acute GVHD spleen cells in vitro. Acute GVHDspleens were removed from anti-gp39 treated, HIg treated or untreatedanimals. Spleens were red cell depleted and then aliquoted at 1×10⁴ percell well. Irradiated stimulator cells (P815) were added to theappropriate wells. After 7 days CTL assays were performed against theappropriate targets as previously mentioned.

Results

GVHD in mice results in splenomegaly. In the mouse, one of theconsequences of cGVHD is the enlargement of the spleen. It is primarilythe host's own cells that infiltrate and enlarge the spleen, althoughthis is in response to the presence of donor cells (Rolink, A. G. et al.(1983) J. Exp. Med. 158:546). FIG. 5 indicates that at 7-14 days afterthe initiation of cGVHD, spleens contains almost twice the number ofleukocytes compared to mice without cGVHD. Treatment of mice at theonset of cGVHD with anti-gp39 (250 μg/mouse, days 0,2,4, and 6), reducethe number of leukocytes/spleen in cGVHD mice to values that wereidentical to mice without disease.

GVHD-induced hyperproduction of polyclonal Immunoglobulin. It has beenreported that hyper production of Ig occurs in mice with cGVHD due tocognate interactions between donor T cells and B cells (Morris, S. E. etal. (1990) J. Exp. Med 171:503). To determine whether anti-gp39 inhibitshyper Ig production, mice with cGVHD were administered anti-gp39. On day7 or 14, spleens were removed from control and cGVHD mice and the Bcells assayed for the spontaneous production of IgG₁ and IgA in vitro.Splenocytes from mice with cGVHD produced high levels of IgA and IgG₁(FIGS. 6A and 6B) in vitro. However, splenocytes from mice with cGVHDand treated with anti-gp39 (days 0, 2, 4 and 6) produced levels of IgG₁and IgA identical to mice without disease. The addition of anti-gp39 tocultures of spleen cells from mice with cGVHD did not reduce the levelsof in vitro Ig production, suggesting that anti-gp39 was exerting itseffects in vivo.

When Hamster Ig (HIg) was used as a control for these experiments it wasfound that it showed no inhibitory role in the induction of GVHD butactually accentuated the disease not only in terms of polyclonal Igproduction but also in the resultant spenomegaly. An 8-fold increase wasdetected in the level of Ig produced by 1 week HIg treated GVHD-inducedmice compared to untreated 1 week GVHD-induced mice. It was apparentthat the HIg was in itself an immunogen. Consequently it was decided todesignate the untreated F1 recipient mice as the relevant control group.

Effect of anti-gp39 on GVHD-induced serum Hyper-IgE and anti-DNAautoantibodies. The course of cGVHD can be monitored by the elevation inserum IgE and antibodies to double stranded DNA (Morris, S. E. et al.(1990) J. Exp. Med. 171:503). Levels of serum IgE were measured using anIgE specific ELISA. Chronic GVHD induced mice were treated withanti-gp39 on days 0, 2, 4 and 6 and then no further antibody wasadministered. Mice were bled at weekly intervals and the levels of serumIgE ascertained (FIG. 7A). cGVHD induces a 10-15 fold increase in serumIgE levels. Administration of anti-gp39 inhibited cGVHD-inducedincreases in serum IgE for up to eight weeks after the initiation ofdisease. In addition to the inhibition of elevated serum IgE,administration of anti-gp39 also blocked the generation of serumanti-DNA autoantibodies. Chronic GVHD induces a 5-10 fold increase inthe levels of anti-DNA antibodies found, which was reduced by anti-gp39treatment (FIG. 7B).

Previous studies have shown that the half-life of anti-gp39 (MR1 clone)is 12 days (Foy, T. M. et al. (1993) J. Exp. Med. 178:1567-1575). ELISAassays specific for the detection of anti-gp39 indicate that theanti-gp39 was undetectable in the serum of treated mice eight weeksfollowing therapy. Therefore, persistent anti-gp39 cannot account forthe protracted suppression of cGVHD. Transfer studies were performed toinvestigate if splenocytes from mice with cGVHD and those that have beenadministered anti-gp39 could transfer cGVHD. Splenocytes from mice givencGVHD and anti-gp39 were incapable of inducing heightened serum levelsof IgE and anti-DNA autoantibodies upon adoptive transfer; whereassplenocytes from mice with cGHVD induced enhanced sIgE and anti-DNAantibodies. These data suggest that the alloreactive T cells have beeninduced to become unresponsive as a result of anti-gp39 treatment.

Detection of alloreactive donotrcells. When cGVHD induced mice wereanalysed for the presence of donor derived cells within their spleens,it was found that compared to normal BDF₁, which stain double positivefor H-2K^(b) and H-2D^(d), cGVHD possessed approximately 5-7 % donorcells. These cells are DBA/2 derived and therefore stain single positivefor H-2D^(d). These cells were detected irrespective of anti-gp39treatment. This illustrate the point that anti-gp39 treatment allowsengraftment of the donor cells without illiciting any of the helperfunction for the recipient B cells that results in polyclonal Igproduction in the untreated cGVHD group.

Effect of anti-gp39 treatment on the induction of acute GVHD. Acute GVHDis associated with the induction of increased anti-allogeneic CTLresponse. While it is clear that gp39-CD40 interactions are critical forT_(h) -B cell interactions, it is not clear if the induction of otherimmune effector mechanisms may also be altered by anti-gp39 therapy.AGVHD was induced by the administration of C57BL/6 spleens to the F₁recipients. As shown in FIG. 8, 12 days following the transfer ofallogeneic cells, a robust H-2^(b) anti H-2^(d) CTL response ismeasured. Treatment of mice with anti-gp39, but not HIg, prevented thegeneration of H-2^(b) anti-H-2^(d) CTL (FIG. 8). In 1 of 2 experiments,treatment with anti-gp39 reduced the CTL responses below that which wasobserved with naive spleen cells. This suggested that spleen cells froma GVHD mice when challenged with. If these spleen cells are thencultured for 7 days in the presence of irradiated P815 cells and then aCTL assay performed these cells fail to mount a secondary stimulation tothe P815 cells, however the normal BDF₁ spleen mount a primary response.The untreated aGVHD induced mice mount a secondary immune response asexpected. These results indicate that the treatment of acute GVHD micewith anti-gp39 makes the CD8+population unresponsive to a secondarychallenge.

Discussion

Reversal of splenomegaly (FIG. 5), inhibition of hyper Ig production(FIGS. 6A and 6B), inhibition of serum levels of IgE and anti-DNAautoantibody production (FIGS. 7A and 7B) in GVH-induced mice byanti-gp39 administration, suggests that anti-gp39 blocked the ability ofthe grafted T cells to induce host B cell activation. This inhibition ofspenomegaly and polyclonal IgG₁ and IgA production remains low 7 daysafter termination of antibody administration. Reduction in IgE andanti-DNA antibodies persists for 8 weeks even when the treatment isterminated. These results indicate that T cell function has beenaffected either by clonal deletion of the reactive T cells or that Tcell anergy has occurred. It has previously been shown (Van den Eertweghet al. (1993) J. Exp. Med. 178:1555-1565) that levels of cytokineproducing cells remain normal in antibody treated mice in in situstudies of immunized mice. This indicates that T cells are not deletedas a consequence of the treatment. When cGVHD mice were analysed for thepresence of donor derived cells they are found to be present whether theanimal is untreated or treated with anti-gp39. Anti-gp39 thus has theability to induce an unresponsive state upon these donor T cells,illiciting inhibition of antibody responses. Indeed, if spleens fromthese animals are transferred into naive recipients levels of antibodyare elevated when the donor spleens are untreated cGVHD but anti-gp39treated cGVHD mice are unable to mount a secondary cGVHD state upontransfer. This data suggests that anti-gp39 interferes with the abilityof T cells to elicit a strong GVHD clinical imnmunopathology andsplenomegaly that the antibody administration elicits unresponsivenesson the CD4⁺ subpopulation.

It has been reported that B cells are required to provide help for theinduction of CTLs as seen by studying B cell deficient mice for theinduction of protective T cell immunity to a Friend murine leukemiavirus-induced leukemia (Schultz, K. R. et al. (1990) Science 249). Itthus seems conceivable that in a aGVHD induced mice, anti-gp39 inhibitsthe activation of B cells and thus prevents antigen presentation andthus prime the CD8⁺ T cells. It has also been suggested that generationof CD8+CTL required interaction with class II-restricted Th cells (vonBoehmer, H. et al. (1979) J. Exp. Med. 150:1134; Keene, J. et al. (1982)J. Exp. Med 155:768) and that when TCR affinity is low, CD4+mediated Tcell help is required in vivo (Gao, X. et al. (1991) J. Immunol.147:3268).

Studies have been performed looking at the role of CD28-B7/BB1interactions in the induction of CD8+CTL responses. It was found thatCD28-B7/BB1 interactions were necessary and sufficient for thegeneration of class I MHC-specific CTL (Harding, F. A. and Allison, J.P. (1993) J. Exp. Med. 177:1791). It has been suggested that the ligandfor CD40 may be an important inducer for B7 (Ranheim, E. A. and Kipps,T. J. (1993) J. Exp. Med. 177:925) as shown by studies involving theinhibition of B7 expression on normal and leukemic B cells by antibodiesto CD40. Together, these studies indicate that anti-gp39 may blockinteraction of CD4+T cells with B cells thus failing to induce theexpression of B7 that allows a B cell to efficiently activate a T cellto proliferate and produce cytokines. Taken together, this data suggeststhat CD4+T cells are required for the induction of CTL formation firstby T- B cells cognate interaction between gp39 and its ligand CD40.Signaling of CD40 on the B cells then allows upregulation of B7/BB1.Reciprocal interaction of B7/BB1 with its ligand CD28 on the T cellsthen allows enhanced T cell proliferation and cytokine production. If,however, only one signal is provided to the T cells, i.e., gp39interaction with CD40, and the second signal is not obtained, then anunresponsiveness is induced in the T cells and it becomes tolerised oranergised.

These studies indicate that when aGVHD is induced in mice, ananti-H-2^(d) response is obtained. However, if the animals are treatedwith anti-gp39, then no CTL response is observed. It may be apparentthat anti-gp39 is inhibiting the CTL formation by a method previouslydescribed (Schultz, K. R et al. (1990) Science 249). The B cells fail tobe activated via CD40 ligation and are thus unable to promote theinduction of CTLs. Furthermore, our studies show that when spleen cellsare challenged in vitro with P815 cells, the cells that were exposed toanti-gp39 in vivo were found unable to mount a secondary anti-H-2^(d)CTL. Thus, this may indicate that anti-gp39 has induced a state oftolerance on the T cell compartment since gp39 is unable to engage CD40,there thus is no B7/BB1 upregulation and so the T cells do not getfurther activated and remain unresponsive. It is also known that restingB cells generally are ineffective stimulators of allogeneic T cells inthe mixed lymphocyte reaction unless preactivated by anti-IgMantibodies, PMA or LPS (Inaba, K. and Steinman, R. M. (1989) J. Exp.Med. 160:1717; Metley, J. P. et al. (1989) J. Exp. Med. 169:239;Frohman, M. and Cowing, C. (1985) J. Immunol. 134:2269). In addition,soluble monomeric antigen directed to B cells for presentation in vivomay result in specific T cell anergy (Eynon, E. E. and Parker, D. (1992)J. Exp. Med. 175:131). Thus, it seems evident, depending on the methodof administration of antigen and APCs involved, that anergy or tolerancemay be induced. Upon challenge of P815 cells to the CD8+T cellscompartment of the spleen, B cells are not required for antigenpresentation, thus alloantigen can be presented directly and CTLinduced. The unresponsiveness of the spleens to secondary stimulationindicates that allospecific tolerance has been induced in this system.

This in opposition to previous results (Foy, T. M. et al. (1993) J. Exp.Med. 178:1567-1575) which indicate that tolerance is not induced by theantibody in an antigen specific system. The two systems differ since theaGVHD model presents alloantigen already bound to antigen presentingcells, whereas with antigen specific systems the antigen is administeredand in vivo is taken up, processed and presented by professional APC. Itthus seems that anti-gp39 may have different effects depending on theantigen being used and the method of presentation.

It can be concluded that anti-gp39 may induce allospecific tolerance inboth the CD4+ and CD8+ compartments of the immune system and this may beobvious beneficial therapeutic intervention when considering transplantimmunology and immunotherapy. It is conceivable that for treatment ofpatients undergoing bone marrow transplants that anti-gp39 therapy willbe sufficient for induction of tolerance to the graft and prevent theinduction of such consequences of transplant treatments as GVHD.

EXAMPLE 6 Production and Characterization of Anti-gp39 Antibodies

Experiment 1--Antibodies directed against human gp39

For induction of antigen-specific T cell tolerance in a human subject,it is preferable to administer an antibody directed against human gp39.The following methodology was used to produce mouse anti-human gp39monoclonal antibodies. Balb/c mice were immunized with a soluble gp39fusion protein, gp39-CD8, in Complete Freund's Adjuvant (CFA). Mice weresubsequently challenged 6 weeks later with soluble gp39-CD8 inIncomplete Freund's Adjuvant (IFA), Soluble gp39-CD8 was given insoluble form 4 weeks after secondary immunization. Mice were thenboosted with activated human peripheral blood lymphocytes 2 weeks later,followed by a final boost with soluble gp39-CD8 after an additional 2weeks. Splenocytes were fused with the NS-1 fusion partner on day 4after final immunization as per standard protocols.

Clones producing anti-human gp39 antibodies were selected based on amultiple screening process. Clones were initially screened by a platebinding assay using gp39-CD8. Positive clones were then screened againsta control CD8 fusion protein, CD72-CD8. Clones which scored positive onthe CD8-CD72 plate binding assay were eliminated. The remaining cloneswere subsequently screened on resting and 6 hour activated humanperipheral blood lymphocytes (PBL) by flow cytometric analysis.Hybridomas staining activated, but not resting, PBL were consideredpositive. Finally, the remaining clones were tested for their ability toblock the binding of CD40Ig to plate bound gp39.

Approximately 300 clones were initially screened against gp39-CD8 andCD72-CD8 in the plate binding assays. Of those clones, 30 were found todetect plate-bound gp39 and not CD8. These clones were subsequentlyscreened for detection of gp39 on activated human PBL. Approximately 15clones detected a molecule on activated PBL, but not resting cells.Specificity was further confirmed by determining the capacity of theclones to block CD40Ig detection of plate-bound gp39. 3 of 10 clonestested block CD40Ig binding in this assay. These clones were 3E4, 2H5and 2H8. Such clones are preferred for use in the methods describedherein. Clones which tested positive on activated, but not resting PBL,were also screened for reactivity with an activated rat T cell clone,POMC8. The clone 2H8 expressed crossreactivity with this rat T cellline.

Experiment 2--Antibodies directed against human gp39

A similar immunization procedure to that described in Experiment 1 wasused to produce additional antibodies directed against human gp39. OneBALB/C mouse was immunized with soluble gp39-CD8 in CFA, followed bychallenge with 6 hour activated human peripheral blood lymphocytes 4weeks later. The mouse was subsequently boosted with soluble gp39-CD8 4days prior to fusion of splenocytes with the NS-1 fusion partner perstandard protocols. Screening of hybridoma clones was performed by flowcytometric staining of 6 hour activated human PBLs. Clones stainingactivated but not resting human PBLs were selected. Six clones, 4D9-8,4D9-9, 24-31,24-43, 89-76 and 89-79, were selected for further analysis.

The specificity of the selected antibodies was confirmed by severalassays. First, flow cytometric analysis demonstrated that all six mnAbsstain activated, but not resting peripheral blood T cells (see FIG. 9Band 9C for a representative example, depicting staining of activated Tcells with 4D9-8 and 4D9-9, respectively). Expression of the moleculerecognized by each of the six antibodies is detectable within 4 hours ofactivation, is maximal between 6-8 hours after activation, and isundetectable by 24 hours after activation. All six mAbs recognize amolecule expressed on activated CD3⁺ PBLs, predominantly of the CD4+phenotype, but a portion of CD8⁺ T cells also express the molecule.Expression of the molecule recognized by the six mAbs is inhibited bythe presence of cyclosporin A in the culture medium, as is theexpression of gp39 (see FIG. 10A and 10B for a representative example,depicting staining of cyclosporin treated T cells with 4D9-8 and 4D9-9,respectively). The kinetics and distribution of expression of themolecule recognized by these mAbs are identical to that of gp39, asdetected by the fusion protein of human CD40Ig. In addition, all sixInAbs block the staining of gp39 by CD40Ig (see FIG. 11A and 11B for fora representative example, depicting inhibition of gp39 staining byCD40Ig in the presence of 4D9-8 and 4D9-9, respectively). In an ELISAassay, all six mnAbs recognize gp39-CD8, a soluble fusion form of thegp39 molecule. Moreover, all six mAbs immunoprecipitate a molecule ofapproximately 36 kd from ³⁵ S-methionine labeled activated human PBLs.The immunoprecipitated molecule is identical to that precipitated by thehuman CD40Ig fusion protein.

The functional activity of the six selected mAbs (4D9-8, 4D9-9, 24-32,24-43, 89-76 and 89-79) was assayed as follows. First, the ability ofthe mAbs to inhibit the proliferation of purified human B cells culturedwith IL-4 and soluble gp39 was measured. Purified human B cells werecultured with gp39 and IL-4 in the presence or absence of purifiedmonoclonal antibodies or CD40Ig at dosages between 0 and 12.5 μg/ml. Bcell proliferation was determined after 3 days in culture by thymidineincorporation. The results (shown in FIG. 12) demonstrate that all sixmAbs can inhibit B cell proliferation induced by gp39 and IL-4. The mAbs89-76 and 24-31 were most effective at inhibiting the induced B cellproliferation. The IC₅₀ (concentration of antibody necessary to inhibitB cell proliferation by 50%) was approximately 1 μg/ml for 89-76 andapproximately 1.25 μg/ml for 24-31.

Next, the ability of the mAbs to inhibit B cell differentiation, asmeasured by Ig production induced by anti-CD3 activated T cells andIL-2, was examined. Purified IgD⁺ human B cells were prepared bypositive selection with FACS and then cultured with anti-CD3 activatedhuman T cells (mitomycin C treated) and IL-2 for 6 days in the presenceor absence of purified anti-gp39 monoclonal antibodies as dosagesbetween 0 and 10 μg/ml. IgM, IgG and IgA production was assessed byELISA on day 6. The results (shown below in Table 3) demonstrate thatall six antibodies can inhibit T cell dependent B cell differentiation,as measured by IgM, IgG and IgA production. The IC₅₀ (concentration ofantibody necessary to inhibit Ig production by 50%) was in the range of1.0 jig/ml to below 0.1 μg/ml for the six mAbs, including the 24-31 and89-76 antibodies.

                  TABLE 3    ______________________________________    Production of Immunoglobulin    mAb        μg/ml IgM        IgG    IgA    ______________________________________    none       --       17,500     6710   4471    4D9-8      0.1      4813       2130   2819               1.0      4394       2558   1519               10.0     1081       389    396    4D9-9      0.1      3594       919    1731               1.0      2659       1233   1606               10.0     374        448    266    24-31      0.1      3863       981    344               1.0      1287       314    165               10.0     1120       596    23    24-43      0.1      6227       4132   432               1.0      3193       2130   192               10.0     7021       1232   1081    89-76      0.1      3783       1069   344               1.0      2180       352    171               10.0     818        551    19    89-79      0.1      9763       1924   3021               1.0      2314       460    156               10.0     183        135    434    ______________________________________

To determine whether the six mAbs recognized distinct epitopes on thehuman gp39 molecule, crossblocking experiments were performed. Activatedhuman PBLs were first blocked with each of the six mAbs (25 μg/ml ofunconjugated antibody). Cells were washed and then stained with 10 μg/mlof biotin-conjugated antibody, followed by reaction with phytoeiythrinconjugated avidin (PE-Av). The staining of the cells with PE-Av wasanalyzed by FACS. The results are shown below in Table 4.

                  TABLE 4    ______________________________________    Blocking            Staining Antibody    Ab      4D9-8   4D9-9    24-31 24-43 89-76 89-79    ______________________________________    none    +++     +++      ++++  ++++  ++++  ++++    4D9-8   ND      -        ++++  ++++  +++   +++    4D9-9   +++     ND       +++   ++++  +++   +++    24-31   +       +        ND    +++   ++    ++    24-43   +       +        +++   ND    ++    +    89-76   +       +        +++   +++   ND    +++    89-79   +       ++       +++   +++   +++   ND    ______________________________________     The intensity of staining and the percentage of positive cells are     represented by the + symbol (++++ = MI > 200; +++ = MI > 125; ++ = MI >     50; + = MI > 25; - no staining above background). ND = not determined.

All antibodies blocked the binding of CD40Ig to activated human PBLs.However, the data shown in Table 4 clearly demonstrate the failure ofsome antibodies to block the binding of other antibodies to activatedhuman PBLs, suggesting that they recognize distinct epitopes on thehuman gp39 molecules.

The 89-76 and 24-31 hybridomas, producing the 89-76 and 24-31antibodies, respectively, were deposited under the provisions of theBudapest Treaty with the American Type Culture Collection, ParklawnDrive, Rockville, Md., on Sep. 2, 1994. The 89-76 hybridoma was assignedATCC Accession Number HB 11713 and the 24-31 hybridoma was assigned ATCCAccession Number HB11712. The24-31 and 89-76 antibodies are of the IgG1isotype.

Experiment 3--Antibodies directed against mouse gp39

In one embodiment of the invention, the gp39 antagonist is an anti-mousegp39 monoclonal antibody, MR1. The following method was used to producethe MR1 monoclonal antibody, and may be used to generate otherantibodies directed toward gp39.

Hamsters were immunized intraperitoneally with 5-10⁶ activated T_(h) 1cells (d1.6) at weekly intervals for six weeks. When the serum titeragainst murine T_(h) 1 was greater than about 1:10,000, cell fusionswere performed with polyethylene glycol using immune hamster splenocytesand NS-1. Supernatant from wells containing growing hybridomas werescreened by flow cytometry on resting and activated T_(h) 1. Oneparticular hybridoma, which produced a Mab that selectively recognizedactivated T_(h) was further tested and subcloned to derive MR1. MR1 wasproduced in ascites and purified by ion exchange HPLC. A hybridoma MR1has been deposited with the American Type Culture Collection andassigned Accession Number HB11048.

EXAMPLE 7 Anti-human gp39 Antibodies can Inhibit Antibody andXenoreactive T Cell Responses

In this example, the effects of antibodies reactive against human gp39on B and T cell reponses were investigated in hu-PBL-scid mice, an invivo model of the human immune system.

Experiment 1--Anti-human gp39 blocks in vivo Tetanus toxoid specificantibody production in SCID mice reconstituted with human PBL

Experiments presented above demonstrated that blockade of gp39 functioninhibited T cell dependent polyclonal Ig production by human B cells invitro (Table 3). To determine whether anti-hgp39 mAb could also inhibitantigen specific B cell antibody production in vivo, anti-gp39 antibodywas adminstered to SCID mice reconstituted with human PBL(C.B-17-scid/scid mice).

C.B-17-scid/scid mice were injected intraperitoneally with 20×10⁶ humanPBL, immunized with 0.25 ml tetanus toxid (TT), and treated with PBS(250 μl) or anti-hgp39 (250 μg/d, twice weekly) and the secondary (IgG)anti-TT antibody response was assessed. Human anti-TT antibody levelswere assessed by antigen-specific ELISA from sera collected weekly.Briefly, polyvinyl microtiter plates were coated with 5 Lf units/ml TTand incubated for 2 hr at 37° C. Plates were washed and subsequentlyincubated for an additional 2 hr with sera samples. A polyclonal goatmouse anti-human IgG (Fab)-alkaline phosphatase antibody used fordetection of TT specific human Ig was added after washing. Plates wereincubated an additional 2 hr at 37° C., developed by reaction withalkaline-phosphatase substrate and the O.D. at 410 nm determined basedon multiple point analysis of sera samples diluted from 1:10-1:10,000.All mice with serum levels of human anti-tetanus toxoid antibody>0.100O.D. at a 1:10 dilution were considered positive. Only positive micewere used in the calculation of the mean±SE values included in thetable. The level of human anti-tetanus toxoid in sera from pre-immunemice or mice not immunized with tetanus toxoid was <0.02 O.D. Data arepresented as mean±SE.

The results are presented in Table 5.

                  TABLE 5    ______________________________________             Anti-Tetanus Antibody (O.D. ± SE)             (Frequency of Mice Containing             Anti-Tetanus Antibody)    Recipient           Treat-  days post immunization    Strain ment    7d        14d     21d     28d    ______________________________________    C.B.-17           PBS     <0.02 (0/10)                             .230 ± .042                                     .224 ± .040                                             .137 +    scid/scid                (7/10)  (8/10)  .007                                             (4/10)           anti-   .162 (1/10)                             <0.02 (0/10)                                     <0.02 (0/10)                                             <0.02           hgp39                             (0/10)    ______________________________________

Immunization of hu-PBL-scid with TT resulted in detectable levels of IgGanti-TT antibody within 14 days post immunization in most animals.However, treatment with anti-hgp39 (24-31; 250 μg/day, twice weekly)completely ablated the secondary anti-TT antibody response in 9/10 miceexamined, demonstrating that in vivo blockade of gp39 function alsoresulted in inhibition of antigen specific humoral responses.

Experiment 2--Anti-hgp39 treatment inhibits xenoreactive T cellresponses

To determine whether anti-hgp39 antibody has an effect on xenoreactive Tcell responses, hu-PBL-scid mice were treated with anti-hgp39 antibodyand the number of engrafted cells was determined. In this model systemof the human immune system, engraftment of T cells in the mice isgenerally a measure of xenoreactive T cell expansion.

NOD/LtSz-scid/scid mice were injected with 20×10⁶ human PBL and furthertreated with anti-hgp39 antibody at 250 μg per injection twice weeklyfor 4 weeks or with PBS. Engraftment was assessed four weeks later byflow cytometric analysis of human CD45⁺ cells in the spleens ofrecipient mice. NOD/LtSz-scid/scid mice were chosen as the recipientstrain for these experiments as human PBL engraft at low levels inuntreated C.B- 17-scid/scid mice.

The results are summarized in Table 6.

                  TABLE 6    ______________________________________                              % of CD45+            Human             Human     Frequency of    Recipient            Cell              Cells in Spleen of                                        Engrafted Mice    Strain  Source   Treatment                              Recipient Mice                                        (>8% CD45+)    ______________________________________    NOD/LtSz-            PBL      PBS      12.7 ± 4.3                                        5/10    scid/scid            PBL      anti-hgp39                              <2.0      0/10    NOD/LtSz-            spleen   PBS      32.6 ± 14.0                                        4/4    scid/scid            spleen   anti-hgp39                              6.9 ± 0.8                                        3/10    ______________________________________

The results demonstrate high engraftment of human PBL in 50% of controluntreated NOD/LtSz-scid/scid recipient mice, with recipient micecontaining >8% CD45⁺ human cells. In contrast, none of the recipientmice treated with anti-hgp39 (0/10) contained >2% human CD45⁺ cells.Similar results were obtained in experiments examining the engraftmentof human spleen cells in NOD/LtSz-scid/scid mice (Table IV). When spleencells were used as the source of donor human lymphocytes, a higherfrequency of CD45⁺ human cells was observed in the spleens of recipientmice (>8% in 4/4 mice). In addition, although treatment with anti-hgp39significantly decreased the percent of CD45⁺ cells in both spleen andblood of recipient mice, some engraftment was observed (>8% in 3/10mice). Higher engraftment in scid mice which received human spleen cellswas most likely due to the higher cell dose injected. The resultsindicate that, in both experiments, treatment with anti-hgp39 results inreduced engraftment of human lymphocytes in scid mice.

Thus, in vivo administration of anti-hgp39 antibody results insuppression of T cell reactivity to xenoantigen, and indicates thatblockade of gp39 function can be utilized as an immunosuppressive agentfor management of human allograft or xenograft rejection.

Experiment 3--Anti-hgp39 treatment does not inhibit antigen-specific Tcell proliferative response of hu-PBL-scid spleen cells

To determine whether treatment of hu-PBL-scid mice with anti-hgp39altered the responsiveness of antigen-specific T cells in vivo, theproliferative response of spleen cells from hu-PBL-scid mice immunizedwith TT and treated with anti-hgp39 was assessed in vitro in secondary Tcell response assays.

Spleen cells were obtained from TT immunized hu-PBL-scid mice four weeksafter TT immunization and in vivo treatment with PBS (250 μl) oranti-hgp39 (250 μg/d, twice weekly). Spleen cells were cultured for 6days in 96 well microtiter plates at a concentration of 1×10⁵ cells/mlin the presence of 2.5 or 5.0 μg/ml TT or medium alone. Cultures werepulsed with 50 μCi of ³ H-thymidine for the final 24 hr of culture andharvested. Stimulation indices were calculated as follows: SI=cpm TT-cpmmedium alone/cpm medium alone.

Table 7 summarizes the results.

                  TABLE 7    ______________________________________                           Frequency of Responding    Recipient Strain                  Treatment                           Mice    ______________________________________    C.B-17 scid/scid                  PBS      3/10                  anti-hgp39                           5/10    NOD/LtSz-scid/scid                  PBS      5/10                  anti-hgp39                           6/10    ______________________________________

Hu-PBL-scid mice treated with anti-hgp39 responded similarly to in vitrostimulation with TT as did hu-PBL-scid mice which were untreated (5/10vs. 3/10 responding mice). Experiments using NOD/LtSz-scid/scid mice asrecipients yielded similar results (Table 7). Thus, anti-hgp39 treatmentdoes not alter the anti-tetanus T cell proliferative response followingengraftment of human PBL in C.B-17-scid/scid or NOD/LtSz-scid/scid miceimmunized with tetanus toxoid, although anti-TT antibodies wereundetectable in these mice. These data demonstrate that treatment withanti-hgp39 does not result in deletion or functional inactivation ofantigen-specific T cells in hu-PBL-scid mice and support the contentionthat inhibition of TT specific antibody responses by anti-hgp39 is dueto blockade of gp39/CD40 interactions and subsequent B cell responsesrather than T cell inactivation.

In view of the preceding results, administration of anti-human gp39antibodies does not inhibit T cell responses to a specific solubleantigen (e.g., TT), at least under the conditions tested, but does blockxenoreactive T cell responses in a T cell transplant recipient. Theseresults indicate that administration of anti-gp39 antibodies to atransplant recipient (e.g., a bone marrow transplant recipient) wouldlikely block expansion of the T cells population that is allo- orxeno-reactive to host antigens, but likely would not block theresponsiveness of the transplanted T cells to foreign antigen (e.g.,environmental antigens).

EQUIVALENTS

Hybridoma cell lines which secrete the 24-31 and 89-76 antibodies weredeposited on Sep. 2, 1994 in the American Type Culture Collection inRockville, Md. and respectively accorded accession numbers HB11712 andHB11713. These deposits were made in accordance with the BudapestTreaty. Further, all restrictions as to the availability of thesehybridoma cell lines will be irrevocably removed upon issuance of apatent to this application.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. The contents of allreferences and published patent applications cited throughout thisapplication are hereby incorporated by reference.

We claim:
 1. A method for inducing T-cell non-responsiveness to atransplanted tissue or organ comprising administering an effectiveamount of an anti-human gp39 (CD40L) monoclonal antibody or fragmentthereof that binds gp39 (CD40L) which antibody or fragment binds theepitope recognized by a monoclonal antibody selected from the groupconsisting of 24-31 secreted by hybridoma 24-31 accorded ATCC AccessionNo. HB1712 and 89-76 secreted by hybridoma 89-76 accorded ATCC AccessionNo. HB1713.
 2. The method of claim 1, wherein said antibody is achimeric antibody containing a non-human variable region and a humanconstant region.
 3. The method of claim 1, wherein said antibody is ahumanized antibody.
 4. The method of claim 1, wherein said organ ortissue is xenogeneic.
 5. The method of claim 1, wherein said organ ortissue is allogeneic.
 6. A method for preventing or treatinggraft-versus-host disease in a bone marrow recipient comprisingadministering an effective amount of an anti-human gp39 antibody (CD40L)monoclonal antibody or fragment thereof that binds gp39 (CD40L) whichantibody or fragment binds the epitope recognized by a monoclonalantibody selected from the group consisting of 24-31 secreted byhybridoma 24-31 accorded ATCC Accession No. HB1712 and 89-76 secreted byhybridoma 89-76 accorded ATCC Accession No. HB1713.
 7. The method ofclaim 6, wherein said antibody is a chimeric antibody containing anon-human variable region and a human constant region.
 8. The method ofclaim 6, wherein said antibody is a humanized antibody.